Subtraction hybridization as commonly used in association with cloning of cDNA derived from mRNA extracted from particular cells that are under investigation is most useful, as already indicated, for developing or producing hybridization probes that can be utilised as screening agents to detect or locate DNA, in clone colonies or cDNA libraries for example, related to genes that are differentially expressed as compared with genes of other cells that exhibit different gene expression characteristics. This technique may, for example, be used in cancer research for comparing the gene products of tumour tissue cells with those of corresponding normal tissue cells in order to study the genetic changes that have occurred at the nucleic acid level. Probes obtained using this technique which are specific to DNA whose expression characteristics are modified by such genetic changes may be useful not only for carrying out genetic screening in connection with cDNA cloning, but also as diagnostic tools.
In a typical procedure for applying this technique of subtraction hybridization to investigate differences in the active genes of a certain sample of test or target cells, e.g. from tumour tissues, as compared with the active genes of a sample of reference cells, e.g. cells from corresponding normal tissue, total cell mRNA is extracted (using conventional methods) from both samples of cells. The mRNA in the extract from the test or target cells is then used in a conventional manner to synthesise corresponding single stranded cDNA using an appropriate primer and a reverse transcriptase in the presence of the necessary deoxynucleoside triphosphates, the template mRNA finally being degraded by alkaline hydrolysis to leave only the single stranded cDNA. In one particular version of the technique, especially relevant to the present invention, care is taken to avoid unwanted synthesis of any second strand cDNA in this initial stage. The single stranded cDNA thus derived from the mRNA expressed by the test or target cells is then mixed under hybridizing conditions with an excess quantity of the mRNA extract from the reference (normal) cells. The latter is herein generally termed the subtraction hybridization "driver" since it is this mRNA or other single stranded nucleic acid present in excess which "drives" the subtraction process. As a result, cDNA strands having common complementary sequences anneal with the mRNA strands to form mRNA/cDNA duplexes and are thus subtracted from the single stranded species present. The only single stranded DNA remaining is then the unique cDNA that is derived specifically from the mRNA produced by genes which are expressed solely by the test or target cells.
From this point onwards, to complete the subtraction process and use the single stranded unique cDNA, for example for producing labelled probes that may perhaps then be used for detecting or identifying corresponding cloned copies in a cDNA clone colony (labelling of such probes is frequently introduced by using labelled deoxynucleoside triphosphates in synthesis of the cDNA), it has hitherto generally been necessary first physically to separate out the common mRNA/cDNA duplexes, using for example hydroxyapatite (HAP) or (strept)avidin-biotin in a chromatographic separation method, after which one or more repeat rounds of the subtraction hybridization may be carried out to improve the extent of recovery of the desired product.
This same need physically to separate out the duplex molecules generated in the subtraction hybridization stage has moreover remained even in many variations or modifications that have been used or proposed in respect of the basic subtraction hybridization scheme outline above, for example variations or modifications in which cDNA derived from mRNA of both cell sources is first synthesized and possibly amplified by a cloning or polymerase chain reaction (PCR) procedure, followed by using one of the cDNA mixtures (after denaturation where the cDNA is double-stranded) as the "driver" for carrying out the subtraction hybridization. The known and published methods for separating out the duplex molecules from the single stranded unique cDNA product, such as the above-mentioned hydroxyapatite or (strept)avidin-biotin chromatographic separation methods, however, are not entirely satisfactory, often leading to incomplete separation of the duplexes and a significant loss of unique cDNA or potential probe material. Such defects can be especially serious when the genetic material of interest provides only mRNA transcripts at a low abundance. Also, these known methods usually involve a fairly complex procedure requiring considerable experimental manipulation throughout, and particularly in producing radiolabelled probes the handling of radioactive material throughout a number of different stages introduces additional complications and hazards.